Mitochondrial DNA (mtDNA) molecules of domestic rats are of two kinds in regard to their sensitivity to cleavage by the bacterial restriction enzyme EcoRI: the mtDNA molecules of some rats contain 4 EcoRI sites (4ES) while the mtDNA molecules of other rats contain the same 4 sites plus two extra sites (6ES). MtDNA molecules from wild rats contain 5 of the EcoRI sites (5ES) found in 6ES mtDNA. Also, differences in sensitivities to the restriction enzymes HhaI, HaeIII and AluI are found among 4ES, 5ES and 6ES molecules at a total of 5 sites. It is now proposed to compare the sensitivities to other restriction enzymes of 4ES, 5ES and 6ES mtDNA molecules (and any oter variants which are found) using agarose gel electrophoresis. We will clone, in bacteria, restriction fragments of 4ES, 5ES and 6ES mtDNA molecules and use the cloned fragments 1) to continue constructing maps of sites which differ in sensitivity to restriction enzymes, 2) to sequence the nucleotides in corresponding regions of molecules which differ in sensitivity to restriction enzymes in order to determine the extent of nucleotide differences in these regions, 3) to map, using RNA-DNA hybridization, the regions of the mtDNA molecule which are transcribed in order to learn whether sites which differ in sensitivity to restriction enzymes are within or between transcribed regions. Further, tissue culture cells containing 6ES mtDNA will be fused with cells containing 4ES mtDNA and an attempt made, using restriction enzyme analysis, to identify and quantitate recombinant mtDNA molecules. Identification of recombinant intermediates in electron microscope preparations of mtDNA crosslinked with trimethylpsoralen will also be attempted in order to elucidate the mechanism by which exchanges are made. The products of mixed fusion between tissue culture cells containing 6ES and 4ES mtDNA will be cloned and the mtDNA analyzed to compare survival of 4ES and 6ES and recombinant mtDNA molecules under the influence of 6ES and/or 4ES nuclei. Finally using 32P-end-labeling of EcoRI fragments of mtDNA derived from progeny of crosses between 6ES and 4ES rats, we will try to determine to what extent the mtDNA of an organism could be derived from the male parent.